The invention relates to novel GPCR-like nucleic acid sequences and proteins. Also provided are vectors, host cells, and recombinant methods for making and using the novel molecules.
G-protein coupled receptors (GPCRs) constitute a major class of proteins responsible for transducing a signal within a cell. GPCRs have three structural domains: an amino terminal extracellular domain, a transmembrane domain containing seven transmembrane segments, three extracellular loops, and three intracellular loops, and a carboxyl terminal intracellular domain. Upon binding of a ligand to an extracellular portion of a GPCR, a signal is transduced within the cell that results in a change in a biological or physiological property of the cell. GPCRs, along with G-proteins and effectors (intracellular enzymes and channels modulated by G-proteins), are the components of a modular signaling system that connects the state of intracellular second messengers to extracellular inputs.
GPCR genes and gene-products are potential causative agents of disease (Spiegel et al (1993), J. Clin. Invest. 92:1119-1125; McKusick et al., J. Med. Genet. (1993) 30:1-26). For example, specific defects in the rhodopsin gene and the V2 vasopressin receptor gene have been shown to cause various forms of retinitis pigmentosum (Nathans et al. (1992) Annu. Rev. Genet. 26:403-424), and nephrogenic diabetes insipidus (Holtzman et al. (1993) Hum. Mol. Genet. 2:1201-1204). These receptors are of critical importance to both the central nervous system and peripheral physiological processes. Evolutionary analyses suggest that the ancestor of these proteins originally developed in concert with complex body plans and nervous systems.
Metabotrophic (P2Y) receptors form a distinct subset of G-protein coupled receptors, whose ligands comprise adenosine 5xe2x80x2-triphosphate (ATP) and/or related nucleotides. P2Y receptors are generally distinguished pharmacologically by the rank order of effectiveness of agonists: some prefer pyrimidines to purines. More than eleven P2Y receptors have been reported. For a review see, for example, North et al. (1997) Current Opinion in Neurobiology 7:346-357. Presently, four subfamilies have been distinguished: 1) P2Y1, 2) P2Y2/P2Y4/P2Y8, 3) P2Y3/P2Y6, and, 4) P2Y5. The P2 receptors may be coupled through G-proteins to signaling pathways involving phospholipase C (which increases inositol-1,4,5-trisphosphate and diacylglycerol formation), phospholipase A2 (with consequent generation of eicosanoids), or adenylate cyclase (which increases cAMP levels). Some members of this receptor family have also been shown to mediate their signals through the inhibition of adenylate cyclase, the inhibition of N-type Ca2+ channels, and the activation of K+ channels. For a review see Boarder et al. (1995) Trends Pharmacol Sci 16:133-139 and Neary et al. 1996 Trends Neurosci 19:13-18.
Endogenous ATP as well as the uracil congener, UTP, act as extracellular signaling molecules and mediate some of their effects via interactions through various members of the P2Y receptor family. Signal transduction through P2Y receptors has recently been implicated in the modulation of neuronal cell membrane ion channels. P2Y type receptors in the rat cerebella or hippocampal neurons (Ikeuchi et al. (1996) Biochem Biophys Res Commun 218:67-71) and guinea pig atrial cells (Matsuura et al. (1996) J Physiol 490:659-671) have been linked to the activation of K+ channels. This coupling is G-protein mediated and membrane-delimited, thus adding P2Y receptors to the family of seven-transmembrane receptors known to act in this way. Furthermore, nucleotides inhibit endogenous Ca2+ currents in neuroblastoma hybrid cells, which contain the P2Y2 receptor. The direct involvement of P2Y2 receptors in this cellular event has since been demonstrated through a direct approach. Rat sympathetic neurons have no native response to UTP. Microinjection of P2Y2 receptor cRNA into the rat sympathetic neurons resulted in expression of the P2Y2 receptor and an acquired UTP-mediated inhibition of their N-type Ca2+ channels (Chen et al. (1996) Endocrinology 137:1833-1840 and Nicholas et al. (1996) Mol Pharmacol 50:224-229). Since P2Y2 receptors are also known to activate phospholipase C, these results indicate that P2Y2 receptors can mediate signal transduction through two independent pathways, depending on the cellular environment.
In P2Y1-transfected COS-7 cells, agonists produce a transient increase in internal Ca2+ associated with the formation of inositol-1,4,5-trisphosphate (Simon et al. (1995) Eur J Pharmacol 291:281-289). P2Y1 receptors have been cloned from turkey brain, mouse and rat insulinoma cells, bovine aortic endothelia cells, rat brain, and human erythroid leukemia cells. Interestingly, the rat P2Y1 receptors are expressed in endothelial cells (B10) isolated from the blood brain barrier. Various nucleotides have been found to mobilize Ca2+ in these cells (Webb et al. (1996) J Pharmacol 119:1385-1392). However, the Ca2+ mobilization response is associated not with an increase in inositol-1,4,5-trisphosphate but rather with the inhibition of adenylate cyclase. This is in contract to the signal transduction mediated by turkey (Filtz et al. (1994) Mol Pharmacol 46:8-14), chicken (Simon et al. (1995) Pharmacol Toxicol 76:302-307) or human (Schachter et al. (1 996) Br J Phrmacol 118:167-173) P2Y1 receptors which when expressed heterologously, clearly induced only inositol-1,4,5-trisphosphate formation. Hence, the P2Y1 receptor, while commonly coupled to phospholipase C, can in some native cells be coupled instead through the G0/Gi cyclase inhibitory pathway.
As extracellular signaling molecules, ATP and UTP are involved in various physiological and pathophysiological processes that have been associated with P2Y receptor family members. The role of ATP in tissue homeostasis, fast excitatory neurotransmission, tissue development, pain transmission, macrophage apoptosis, platelet aggregation, astroglia cell function, and the development and maturation of the nervous system has been established and current evidence indicates that the P2Y receptors mediate many of these cellular effects via an interaction with the extracellular ATP ligand. See for example, Burnstock et al. (1996) Drug Dev. Res. 39:204-242 and Williams et al (1999) Progress in Brain Research 120:93-106. Furthermore, UTP, acting via the P2Y receptors, is a potent and selective modulator of mucocilliary transport. Thus, members of the P2Y receptor family mediate cellular responses produced by extracellular ATP, UTP, and/or related nucleotides (Anderson et al. (1997) Trends Pharmacol. Sci. 18:387-392).
Extracellular ATP is known to be hyperalgesic: peripheral administration of ATP produces pain and ATP can enhance the production of prostaglandins that also produce pain (Needleman et al. (1974) Circ. Res. 34:455-460). Burnstock ((1996) Autonomic Neuroscience Institute, sheet 8) has suggested that locally produced ATP may contribute to the pain associated with causalgia, reflex sympathetic dystrophy, migraine, angina, lumbar, pelvic and cancer pain. Salter et al. have shown that P2Y receptors in dorsal horn astrocytes respond to ATP by increasing Ca2+ levels and thus, P2Y receptors may be involved in mediating the pain response (Salter et al. (1994) J. Neurosci. 14:1563-1575). During reactive-hyperemia, large amounts of ATP are released from vascular endothelial cells that act on P2Y receptors, resulting in the release of nitric oxide and vasodilatation. Burnstock proposed that in the microcirculation, ATP diffuses from the endothelial cells to activate nociceptive endings of sensory nerve fibers in the adventitia (Burnstock (1996) Lancet 347:1604-1605). ATP released from platelets during aggregation, which has been reported to increase in migraine, may also contribute to the initiation of pain via nociceptive receptors. Consistent with this hypothesis is the finding that nociception from blood vessels is independent of the sympathetic nervous system under physiological conditions in humans (Kindgen et al. (1997) Cur J. Pharmacol 328:41-44).
Tumor cells are also known to contain exceptionally high levels of ATP. It has been suggested that when the tumor reaches a size that leads to breakage of cells during abrasive movements, the ATP released acts on nociceptive endings of sensory nerves in the vicinity, resulting in the sensation of pain. Furthermore, ATP released from damaged muscle following major accidents or surgery could be involved in local pain and in pain associated with traumatic shock. In addition to the hyperalgesic actions of ATP, the nucleotide has also been characterized as a messenger for innocuous mechanical stimuli and P2Y1 receptors have been suggested to mediate tactile responses in sensory neurons.
Following pathological events in the brain tissue, purine nucleotides and nucleosides are released into the extracellular space. The (patho)physiological importance and molecular mechanisms of the purinoceptor-mediated effects are unknown. It has been suggested that following ischemic and other brain injuries, ATP play s a trophic role in the initiation and maintenance of reactive astrogliosis. Franke et al. (1999) Glia 28:190-200 found that the P2 antagonist, 2-MeSATP, stimulates gliosis following CNS injury. Specifically, the P2 antagonist reduced astroglial proliferation after stab wound as well as after application of 2-MeSATP. These results suggest the involvement of P2 receptors in the generation of astrogliosis in vivo.
The ability of high doses of ATP to evoke tonicclonic convulsions implicates the nucleotide in fast excitatory neurotransmission and thus, ATP may play a role in seizure generation (Buday et al. (1961) J. Pharmacol 8:2221-2228). Furthermore, ATP may be a prime mediator of neurogenic inflammation via its actions on P2 receptors on neutrophiles, macrophages and monocytes leading to cytokine and prostaglandin release (Dubyak et al. (1993) Am. J. Physiol. 265:C577-C606 and Needleman et al. (1974) Circ. Res. 34:455-460). In this context it is of interest that the putative P2Y7 receptor cloned from human erythroleukemia cells was subsequently found to be the leukotriene B4 receptor, suggesting that the receptors for prostanoids and ATP may be functionally related.
Perilymphatic ATP, likely acting via P2Y receptors can depress the sound-evoked gross compound action potential of the auditory nerve and the distortion product otoacoustic emission, the latter a measure of the active process of the outer hair cells. (Kujawa et al. (1994) Hear Res. 76:87-100). P2Y receptor expression is also observed in the marginal cells of the stria vascularis, a tissue involved in providing the ionic and electrical gradients of the cochlea. Little is currently known about the pharmacology and receptor associated physiology of hearing and vestibular function, however, it has been suggested that ATP may regulate fluid homeostasis, hearing sensitivity, and development.
Furthermore, ATP and UTP are potent stimulants of chloride secretion in airway epithelium (Mason et al. (1993) Am. Rev. Respir. Dis. 147:A27) and mucin glycoprotein release from epithelial goblet cells (Lethem et al. (1993) Am. J. Respir. Cell. Mol. Bio. 9:315-322) acting via the P2Y2 receptor. P2 receptors may therefore play a role in the ability of UTP to stimulate mucociliary clearance and sputum expectoration in smokers, non-smokers and patients with chronic bronchitis. ATP also appears to have a potential role in asthma and ATP and UTP have been demonstrated to potentiate IgE-mediated mast cell histamine release. Evidence indicates such effects-may be mediated by P2Y receptors (Schulman et al. (1998) Drug Dev Res 43:40).
Accordingly, GPCRs are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown GPCRs. The present invention advances the state of the art by providing novel seven-transmembrane proteins/GPCRs.
Isolated nucleic acid molecules corresponding to GPCR-like nucleic acid sequences are provided. Additionally, amino acid sequences corresponding to the polynucleotides are encompassed. In particular, the present invention provides for isolated nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO:2 or the nucleotide sequences encoding the DNA sequence deposited in a bacterial host as ATCC Accession Numbers PTA-2365, PTA-2166, and PTA-2366. Further provided are GPCR-like polypeptides having an amino acid sequence encoded by a nucleic acid molecule described herein.
The present invention also provides vectors and host cells for recombinant expression of the nucleic acid molecules described herein, as well as methods of making such vectors and host cells and for using them for production of the polypeptides or peptides of the invention by recombinant techniques.
The GPCR-like molecules of the present invention are useful for modulating pain transmission; immune and inflammatory responses; cell growth, differentiation, and death; and, the release of hormones, neurotransmitters, and cytokines. The molecules are useful for the diagnosis and treatment of neurologic disorders, such as central nervous system or peripheral nervous system disorders, including, for example, epilepsy, schizophrenia, depression and anxiety, Alzheimer""s and Parkinson""s disease, trauma, ischemia, sclerosis, various forms of encephalopathies, and demyelinating diseases; pain disorders or conditions, including, for example, vascular pain, including angina, ischemic muscle pain, migraine, lumbar pain, pelvic pain, and sympathetic nerve activity including inflammation associated with arthritis; and exocrine and endocrine mediated disorders, including for example, disorders of airway electrolyte metabolism, i.e. cystic fibrosis, chronic airway infections, and other lung disorders. Disorders associated with the following cells or tissues are also encompassed: brain, cortex, dorsal root ganglion (DRG) neurons, sciatic nerve, spinal cord, heart, kidney, gastro muscle, liver, lung, and, skin. Additionally, the molecules of the invention are useful as modulating agents in a variety of cellular processes including the mobilization of intracellular molecules that participate in a signal transduction pathway. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding GPCR-like proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of GPCR-like-encoding nucleic acids.
Another aspect of this invention features isolated or recombinant GPCR-like proteins and polypeptides. Preferred GPCR-like proteins and polypeptides possess at least one biological activity possessed by naturally occurring GPCR-like proteins.
Variant nucleic acid molecules and polypeptides substantially homologous to the nucleotide and amino acid sequences set forth in the sequence listings are encompassed by the present invention. Additionally, fragments and substantially homologous fragments of the nucleotide and amino acid sequences are provided.
Antibodies and antibody fragments that selectively bind the GPCR-like polypeptides and fragments are provided. Such antibodies are useful in detecting the GPCR-like polypeptides as well as in regulating pain transmission, nervous system function and development, release of hormones, neurotransmitters and cytokines, immune and inflammatory responses and cell growth, differentiation and death.
In another aspect, the present invention provides a method for detecting the presence of GPCR-like activity or expression in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of GPCR-like activity such that the presence of GPCR-like activity is detected in the biological sample.
In yet another aspect, the invention provides a method for modulating GPCR-like activity comprising contacting a cell with an agent that modulates (inhibits or stimulates) GPCR-like activity or expression such that GPCR-like activity or expression in the cell is modulated. In one embodiment, the agent is an antibody that specifically binds to GPCR-like protein. In another embodiment, the agent modulates expression of GPCR-like protein by modulating transcription of a GPCR-like gene, splicing of a GPCR-like mRNA, or translation of a GPCR-like mRNA. In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of the GPCR-like mRNA or the GPCR-like gene.
In one embodiment, the methods of the present invention are used to treat a subject having a disorder characterized by aberrant GPCR-like protein activity or nucleic acid expression by administering an agent that is a GPCR-like modulator to the subject. In one embodiment, the GPCR-like modulator is a GPCR-like protein. In another embodiment, the GPCR-like modulator is a GPCR-like nucleic acid molecule. In other embodiments, the GPCR-like modulator is a peptide, peptidomimetic, or other small molecule.
The present invention also provides a diagnostic assay for identifying the presence or absence of a genetic lesion or mutation characterized by at least one of the following: (1) aberrant modification or mutation of a gene encoding a GPCR-like protein; (2) misregulation of a gene encoding a GPCR-like protein; and (3) aberrant post-translational modification of a GPCR-like protein, wherein a wild-type form of the gene encodes a protein with a GPCR-like activity.
In another aspect, the invention provides a method for identifying a compound that binds to or modulates the activity of a GPCR-like protein. In general, such methods entail measuring a biological activity of a GPCR-like protein in the presence and absence of a test compound and identifying those compounds that alter the activity of the GPCR-like protein.
The invention also features methods for identifying a compound that modulates the expression of GPCR-like genes by measuring the expression of the GPCR-like sequences in the presence and absence of the compound.
Other features and advantages of the invention will be apparent from the following detailed description and claims.